The invention herein relates to measurement of the concentration of antigenic substances, most usually in biological fluids, and to assessing the immunologic competence of a subject organism. More specifically, the invention employs perturbed angular correlation spectroscopy (PAC) to monitor the interaction of antibody/antigen-sensitized vesicles loaded with a gamma-emitting cation with antibodies for the subject antigen/antibodies.
Techniques for immunoassay of specific substances have undergone extensive expansion in the last 10 to 15 years. The concentration of antigen is most commonly sought in biological fluids, for example, blood, urine and spinal fluid, although there is no theoretical reason why antigen concentration could not be measured in the context of any fluid medium, biological or not. Radio immunoassay (RIA) is an extremely sensitive and reasonably specific technique and has found a large scope of use (Thorell et al 1978); however, it requires separation of bound from unbound antigen in order to assess the results. Spin immunoassay (SIA) has also been employed (Leute, et al. 1972, Hsia et al 1973, Wei et al. 1975); it obviates the need for separation, but is relatively insensitive. An additional technique, using enzyme mediated reactions as the measure of the extent of the antigen-antibody reaction has also recently been employed. Again, ordinarily, separation of the bound material is required, although this is not always the case. The present invention, as employed in the measurement of the concentration of the antigenic substances, offers a method with sensitivity comparable to RIA, which does not require separation of the bound antigen.
In addition to measurement of antigen concentration, there is considerable interest in determining the general competence of the immune system of an organism. This is desirable, for example, in order to assess the effects of various environmental conditions on the immune system (Tengerdy, et al. 1972, 1973; Bramen, et al. 1973; Thomas, et al. 1973; Nulsen, et al. 1974; Kripke, et al. 1976). In addition, it is frequently desirable deliberately to manipulate the immune system, and to monitor these manipulations. Protocols and drugs are, for example, employed to immunosuppress recipients of organ transplants to prevent rejection (Maugh, 1980; Trotta, et al. 1981) and thus it is necessary to measure the effectiveness of suppressing immune response. Conversely, assays of immunological competence are necessary to assess the effectiveness of attempts to potentiate the immune system, employing, for example, adjuvants which may help control malignant growth (Schnipper, et al. 1980; Taniguchi, et al. 1981). The present method, in view of its ability to quantify immunological response, is an aid in designing and administering these drugs and protocols which affect the immune system.
The present invention takes advantage of the ability of complement to mediate the lysis of liposomes sensitized with antigen that bind to antibody.
It is known that liposomes loaded with a complexed gamma-emitting cation, usually In-111 linked to a chelator such as nitrilotriacetic acid (NTA) can be assayed for integrity by the use of perturbed angular correlation spectroscopy, (PAC), which generates a factor related to the tumbling rate of the gamma emitter. When the gamma emitter is enclosed in the vesicle, the tumbling rate is greater than when it is freed into a biological fluid, because of the ability of the gamma-emitting cation to associate itself with other proteins found in the fluid. The tumbling frequency is thereby slowed. Accordingly, it has been shown that PAC can be used as a measure of the extent of lysis of liposomes containing such gamma emitters. See, for example, Mauk, M. R. and Gamble, R. C. Proc. Natl. Acad. Sci. (USA) 76: 765-769 (1979).
In the present invention, this technique is used to follow that lysis of the "sensitized" vesicles which is due to specific binding of the vesicle containing an antigenic surface to the antibody correlated with it. When such binding occurs, complement mediates lysis. Accordingly, this technique serves as a method to assess the antigen-antibody reaction in any suitable context.